A. General Background to Cation Channels
Cation channels are a diverse group of proteins that regulate the flow of cations across cellular membranes. The selectivity of a cation channel for particular cations typically varies with the valency of the cations, as well as the specificity of a given channel for a particular cation. Some cation channels display almost no selectivity for cations with the same valence (see, e.g., Saitow et al, Biochim Biophys Acta 1327(1):52–60 (1997)). Other channels are clearly selective for particular cations but are permeable to other cations to varying degrees (see, e.g., Park & MacKinnon, Biochemistry 34(41):13328–33 (1995) and Gauss et al., Nature 393(6685):583–7 (1998)).
Cation channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, transduction of sensory stimuli, and regulation of renal electrolyte transport. Cation channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, sensory, and epithelial tissue. These channels allow the flow of various cations in and/or out of the cell under certain conditions. For example, the inward flow of cations upon opening of these channels makes the interior of the cell more positive, thus depolarizing the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, cyclic nucleotides or other secondary messengers, extracellular ligands, and ATP-sensitivity.
Certain classes of cation channels are formed by four alpha subunits and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). Some cation channels may contain other structurally distinct auxiliary, or beta, subunits. These subunits do not form potassium channels themselves, but instead modify the functional properties of channels formed by the alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify their inactivation kinetics (Heinemann et al., J. Phsyiol. (Lond); 493:625–633; 1996 and Shi et al., Neuron 16(4):843–852, 1996). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al, Nature 384:80–83, 1996).
B. Hyperpolarization-Activated Cation Channels: HAC1 and HAC2.
Specialized cells in the heart and brain can create rhythmic activity due in a large part to a depolarizing mixed sodium/potassium current known as Ih (see, e.g., Santoro et al., Cell 93:717–729 (1998)). This pacemaker current is generated by hypolarization activated channels that are present in the heart (see, e.g., DiFrancesco, Ann. Rev Physiol. 55:455–72 (1993) and brain (see, e.g., Papa, Ann. Rev. Physiol 58:299–327 (1996). In addition to contributing directly to rhythmic activity in the brain and heart, these channels may contribute significantly to resting membrane potentials in neurons and other cell types from a variety of tissues.
Recently a family of hyperpolarization-activated channels, given the acronym HAC, was isolated from mouse (see, Ludwig et al., Nature 393:587–91 (1998)). Ludwig et al. reported isolating three different ion channels (mHAC1, mHAC2 and mHAC3). The mouse HAC proteins are members of the voltage-gated cation channel super family and also have a cyclic nucleotide binding domain capable of binding cAMP and cGMP. Mouse HAC1 exhibits the general properties of Ih and may be responsible for pacemaker activity.
Another group also identified the same gene family, in this instance identified by the acronym BCNG. For instance, the BCNG-1 (HAC2) ion channel was isolated from mouse cells and is expressed in the brain (see, e.g., Santoro et al., Proc. Natl. Sci. USA 94:14815–20 (1997)). The human BCNG-2/HAC1 and BCNG-1/HAC2 have also been cloned (see, e.g., Santoro et al., Cell 93:717–729 (1998)). Since then, several related mouse genes (e.g., BCNG-1/HAC2, partial BCNG2/HAC1, partial BCNG3/HAC4, and partial BCNG4/HAC3) with expression in various tissues, including heart and brain, have been isolated (see, e.g., Santoro et al., Cell 93:717–729 (1998)).
Phylogenetic analysis indicates that mHAC3 is more distantly related to mHAC1 or mHAC2 than are mHAC1 and mHAC2 to each other. Human HAC3 has not been previously isolated. Isolation of human HAC3 is therefore desirable, to better understand the physiology of HAC3 in humans and for the development of therapeutic and diagnostic applications to diseases related to hHAC3 in humans.